Infection caused by influenza B virus remains a serious threat to global healthcare, due to naturally evolving strains and to their potential use as manmade bio-weapons by terrorists. Hemagglutinin (HA) is a major surface glycoprotein involved in four aspects of influenza infection: (a). HA is the target of antibodies for neutralization of infectivity; (b). HA undergoes antigenic drift to escape neutralization; (c). HA binds to cell-surface receptors, sialic acid, to initiate the infection; and (d). HA mediates fusion of viral and target membranes to allow viral entry. One now has extensive knowledge on structure and function of influenza A virus HA (AHA) that was greatly facilitated by knowing the structure of H3 subtype that was solved almost 30 years ago. In marked contrast, influenza B virus HA (BHA) is poorly understood, particularly on antibody neutralization, antigenic drift and receptor binding that differ significantly from those of AHAs. This team has most recently determined the first structures of BHA from influenza B/HongKong/8/73 strain (B/HK HA) in unliganded state as well as in complex with human and avian receptor analogs. These structures have provided a solid foundation to address the three most critical issues of influenza B virus in this proposal: the precise antigenic structure, the low receptor-binding affinity, and the impact of antigenic drift on receptor binding of BHA. The overall hypothesis is that the structural differences between BHA and AHA dictate the functional differences between them, which are further modulated by diversifying positive selective pressure. The specific aims are: (1). Define a new antigenic site in BHA. A more accurate definition of the antigenic structure of BHA will substantially enhance one's understanding of the host-pathogen relationship. (2). Determine the molecular determinants for BHA receptor-binding affinity. The results of this aim will not only reveal the molecular determinants for the receptor-binding affinity, but also may help future rational design of high-affinity receptor analogs that can efficiently block influenza B virus from attaching to host receptors, thus reducing the severity of influenza B virus caused infection. (3). Characterize the impact of natural substitutions at receptor-binding site on BHA antigenicity and receptor binding. The results of this aim will provide an atomic basis for their impacts, and elucidate the degree of variations in receptor binding that BHA can tolerate during antigenic drift. The recent accomplishment of this team in solving the first set of structures of BHA makes it uniquely qualified for the proposed studies. The results from these studies will substantially improve one's fundamental understanding of influenza B virus as an important human pathogen, may help future development of novel strategies for combating morbidity and mortality caused by influenza B virus infection, and provide a strong driving force to the influenza B virology field. PUBLIC HEALTH RELEVANCE: Influenza virus is among the National Institute of Allergy and Infectious Diseases Category C priority pathogens. Infection caused by influenza B virus remains a serious threat to global healthcare. The proposed studies aim to substantially improve the fundamental understanding of influenza B virus as an important human pathogen, may help future development of novel strategies for combating morbidity and mortality caused by influenza B virus infection, and provide a strong driving force to the influenza B virology field.